Metnase is a human protein with a SET (lysine methylase) domain and a Mariner transposase (nuclease) domain. Metnase is implicated in several aspects of DNA dynamics. Metnase promotes integration of DNA in a sequence-independent manner, but it is not known if integration sites are random. Metnase interacts with DNA ligase IV (LigIV) and NBS1, and appears to be an alternative to the well-known LigIV binding partner, XRCC4. LigIV and XRCC4 function in the final step of DNA double-strand break (DSB) repair by non-homologous end-joining (NHEJ). Metnase increases the efficiency and accuracy of NHEJ of plasmid substrates, and therefore appears to augment, or function redundantly with, classical NHEJ factors. Both SET and nuclease domains are required to promote NHEJ. Metnase has no apparent role in DSB repair by homologous recombination, but siRNA knockdown of Metnase suppresses random integration and enhances homology-directed integration (gene targeting). Metnase is not an active transposase as it does not efficiently mobilize endogenous Mariner elements. However, Metnase influences translocations perhaps reflecting its role in NHEJ. Defects in classical NHEJ proteins cause genome instability and predispose to cancer. Metnase is expressed in most human tissues, and Metnase levels are generally highest in proliferating tissues. siRNA knockdown of Metnase slows cell growth by elongating S phase, and sensitizes cells to replication stress induced by hydroxyurea and methylmethane sulfonate. Metnase is phosphorylated after DNA damage on serine 495 (S495), but the responsible kinase is unknown. Metnase interacts with TopoII1 and promotes TopoII1 chromosome decatenation activity. TopoII1 has been implicated in chromosomal translocations, including chemotherapy-induced translocations in secondary tumors. Our central hypothesis is that Metnase influences genome integrity through its roles in NHEJ, DNA integration, and chromosomal translocation. Much of what is currently known about Metnase is based on in vitro and plasmid-based in vivo assays. Here we propose two Specific Aims focused on in vivo chromosomal endpoints that will define the functional significance of the Metnase SET, nuclease, and phosphorylation domains in NHEJ and DNA integration. We will also determine the functional significance of the Metnase-LigIV interaction in NHEJ and integration, and whether Metnase influences chromosome translocations when TopoII1 is inhibited. These projects will provide mechanistic information about Metnase function during chromosomal DSB repair, integration and translocation. This information will provide new insights into (i) cellular stress responses and the maintenance of genome integrity, which relate to cancer etiology and treatment strategies; and (ii) the machinery responsible for DNA integration, which directly regulates genome modification by viral and non-viral DNA insertion, and may also be important for chromosomal translocations in human diseases including leukemias and lymphomas. Mechanistic insights into these processes will foster the development of more effective and safer cancer radio- and chemotherapy protocols, anti-viral agents, and gene therapy systems. PUBLIC HEALTH RELEVANCE: The human protein Metnase functions in DNA double-strand break repair, DNA integration into the human genome, and chromosomal translocations. The proposed studies will provide mechanistic information about cellular functions of Metnase. This information will provide new insights into cellular stress responses and the maintenance of genome integrity, both of which are important for cancer initiation and progression, and for cancer treatment. The proposed studies are also relevant to mechanisms of genome modification (mutagenesis) by viral and non-viral DNA insertion, and chromosomal translocations in human diseases including leukemias and lymphomas. Mechanistic insights into these processes will foster development of more effective and safer cancer radio- and chemotherapy protocols, anti-viral agents, and gene therapy systems.